There are different known apparatus and methods, e.g., ultrafiltration, chromatography, HPLC and the like, for classifying extremely small, charged particles. Unfortunately, problems exist especially with separation of living cells, native DNA, DNA having double strand breaks (dsb), RNA, viruses, proteins, polymer molecules, metal molecule aggregates and powders and the like. The problems associated with classifying DNA molecules and their fragments will be discussed, it being understood that problems exist in classifying other particles.
The measurement of radiation or drug-induced DNA damage is fundamental to many aspects of radiation and medical research because DNA is recognized as the most important target for radiation and some drug-induced cell killing. DNA dsb have been associated with cell killing and other deleterious effects of ionizing radiation. DNA fragments are the result of dsb.
It is desirable to not only study the level of dsb induced by a small, nonlethal dose of radiation or drugs to determine the dose's relevance to cell killing but also induced by a large superlethal dose. A superlethal dose exceeds the dose range studied in cell survival experiments and therefore the cell cannot survive such a dose.
The level of dsb is indicated by the reduction in the average size of the DNA molecules in the test cells as compared to the size of the DNA molecules of untreated control cells. The sizes of the DNA fragments can be measured by the fragments' rates of sedimentation using a sucrose gradient sedimentation assay that includes a centrifugation step, nondenatured (or neutral) filtration through a 0.2 micron filter using a filter elution assay, or by electrophoretic mobility using a Pulsed Field Gel Electrophoresis (PFGE) assay. These assays, based on widely different physical principles, can also be applied to measure the reduced mobility and increased size of DNA fragments due to formation of DNA-DNA or DNA-protein crosslinks.
The sensitivity of these assays (defined as the ability to detect dsb caused by small doses) is in part determined by how the DNA samples had been prepared. For example, sucrose gradient sedimentation assay involves pipeting isolated DNA which causes shear forces that fragment the DNA and gives an unreliable result. Consequently, superlethal doses have to be used to minimize the relative amount of dsb induced by sample preparation. Centrifugation can take up to 96 hours to complete, which is an undesirably long time. It cannot classify the fragments based on size. When cells are lysed in a non-ionic detergent and high salts (above 2M NaCl), the DNA is isolated free of structured proteins, such as histones, and it exists as a long fragile polymer which readily fragments during centrifugation which is undesirable. This assay requires expensive, sophisticated equipment that must be operated by a skilled worker.
PFGE uses electrical fields that periodically change direction instead of a constant field in one direction as occurs in conventional electrophoresis. The resolution of large molecules is primarily accomplished by exploiting the time that the molecule takes to change directions in response to the changing electrical fields. DNA migration is achieved by using a longer pulse time and/or a higher field in the forward direction. A number of different apparatuses have been described based primarily on variations in geometry of the electrical field. Depending on the DNA trapped, PFGE can take from 72 hours up to 10-15 days to perform, which is also an undesirably long time.
In filter elution, a sample is collected for a time period that starts when the first drop is eluted. After the collection time period ends, a second sample is collected. This is repeated for at least 17 hours which is an undesirably long time. The amount collected during each time period is compared to the other amounts collected. The contents of the samples are not analyzed. The smaller the collection time period, the more accurate the assay but the more difficult the sample collecting and the longer it takes to run the assay.
Other problems with these apparatus and methods have been noted, as discussed below.
Difficulties in measurement of dsb has contributed to the lack of definitive understanding of the role of the dsb in cell killing. Blazek et al., Evidence from nondenaturing filter elution that induction of double-strand breaks in the DNA of Chinese Hamster V79 cells by .gamma. radiation is proportional to the square of dose. Radiation Research 119, 466-477 (1989).
The main difficulty lies in detecting dsb in mammalian cells at low doses. Dsb have usually been determined by lysing cells in detergents on the top of neutral sucrose gradients and sedimenting the freed DNA through the gradient to estimate its relative molecular mass. This method has proved suitable for organisms with a small amount of chromosomal DNA such as bacteria or yeast. However, under similar conditions the large amount of DNA of a mammalian cell sediments in an anomalous fashion, even when the DNA is largely freed from membranes and other cellular components, unless large [superlethal] dosages of radiation are given (100 Gy.ltoreq.D.ltoreq.2000 Gy). Thus, molecular studies of dsb induction and repair have usually been carried out at dosages far in excess of those used for cell survival studies. Although other methods have been developed for the measurement of dsb at relatively low dosages, the calibration of these methods requires an absolute measurement of dsb within the same dose range. Blocher, DNA double-strand breaks in Ehrlich ascites tumor cells at low doses of X-rays. I. Determination of induced breaks by centrifugation at reduced speed. Int J. Radiat, Biol., 42:3, 317-328 (1982).
See, Bradley, M. O. and Kohn, K. N., X-ray induced DNA dsb production and repair in mammalian cells as measured by neutral filter elution., Nucleic Acid Research, 7, 793-804 (1979) for additional shortcomings of filter elution.
PFGE is undesirable for use with large DNA molecules, e.g., mammalian DNA that containing 6.times.10.sup.9 nucleotide pairs, that are 55 to 60 centimeters long and therefore have a high axial rotation. This is because it is difficult for the DNA to move through the gel without getting trapped therein. Viovy et al., Irreversible trapping of DNA during cross-field gel electrophoresis. Electrophoresis, 13, 1-6 (1992). For a review of PFGE, see, D. Cantor et al., Pulsed-field gel electrophoresis of very large DNA molecules. Annual Review of Biophysical and Biophysical Chemistry, 17, 287-304 (1988).
Because of the large molecular weight of mammalian DNA, anomalous sedimentation patterns are often observed on sucrose gradients and reliable molecular weights can be determined only at superlethal dosages of ionizing radiation. In the filter elution method, many extrinsic factors affect the rate at which the DNA elutes, e.g., pH of the lysis solutions, buffer composition, position of cells in the cell cycle and lysis conditions. Slow-speed centrifugation improves sensitivity, but is extremely dependent upon correct lysis. Stamato et al., Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells. Radiation Research 121, 196-205 (1990).
Conventional electrophoresis is limited to the separation of DNA molecules below 0.75 megabase pairs (Mbp) because larger DNA molecules orient along the direction of the electrical field and move irrespective of their molecular weight ("reptation"). The orientation also occurs during neutral gradient sedimentation (movement of DNA molecules under the force of a centrifugal field). The elongation of large molecules is not only determined by the laws of hydrodynamics, but is also an unavoidable necessity in agarose gel electrophoresis because the diameter of the gel pores is much smaller than that of the random coil of a DNA molecule, even at low concentrations. Thus, DNA molecules can only migrate through the agarose gel by being elongated and reptating through the chain of gel pores. Blocher et at., CHEF electrophoresis, a sensitive technique for the determination of DNA double-strand breaks International Journal Of Radiation Biology 56:4, 437-448 (1989). Reptation is a problem because it makes the test results inaccurate.
An addition problem with PFGE is DNA trapping in the gel matrix of the specimen or trapping in the gel fibers along the plate in so called "fight knots", which do not permit DNA molecules to move. Tarreel, C. et at., Irreversible Trapping of DNA during Crossed Field Gel Electrophoresis, J. Nucleic Acids Research, 18, 569-575 (1990) and Viovy, J-L. et at., Electrophoresis, 13, 1-6 (1992).
Typically, agarose gel is used in an amount in the range of about 0.75 to about 1 wt %. This concentration results in a gel that hinders the movement of high molecular weight particles therethrough.
About 20% and more of the DNA in a sample that is irradiated with high dosage ionizing radiation is not released from the gel of the sample despite having a large number of dsb. Thus, the accuracy of the assay is questionable.
Radioactive labeling of the DNA is usually required regardless of the apparatus or method used. Unfortunately, radioactive labeling limits the applicability to established cell lines and excludes primary tissues and non-proliferating cells.
Presently, food is being preserved by irradiation. Unfortunately, the above described DNA based assays cannot identify irradiated food.
Thus, it has been recognized that the most of the currently available techniques for measuring DNA dsb are time consuming and relatively expensive, have many shortcomings in reproducibility, sensitivity and applicability to most of the important particles to be studied (particularly chromosomal DNA from mammalian cells). Critical assessment of the available techniques leads to a general agreement that sensitivity of current assays for quantification of DNA dsb is a major problem. Also, all the assays suffer from an inability to produce purified DNA for analysis without inducing additional fragmentation.
An apparatus and method exhibiting the ability to classify particles by size and which do not exhibit the above-identified shortcomings and problems of the known apparatus and method are highly desirable.